Monitoring of Nonacog Beta Pegol: One‐Stage Clotting Assay With Kaolin Reagent as a Practical Alternative to Chromogenic Methods

ABSTRACT

Introduction

Accurate monitoring of nonacog beta pegol (N9‐GP) is essential to ensure appropriate treatment and to avoid under‐ or overdosing, which may result in clinical complications. The product's extended half‐life, achieved through molecular modification, poses challenges for activity measurement, particularly when using a one‐stage clotting assay (OSA). Therefore, a chromogenic substrate assay (CSA) is preferred, although it may be less practical for urgent or routine monitoring.

Aim

To evaluate OSA and CSA for N9‐GP measurement, focusing on the suitability of the OSA STA CK Prest reagent on the STA R MAX analyser.

Methods

Factor IX activity (FIX:C) of N9‐GP in vitro spiked and post‐infusion samples were measured using various OSA reagents and CSA across different analysers. The in vitro sample results were evaluated as percentage recovery; the OSA STA CK Prest results from both spiked and post‐infusion samples were compared with those obtained by the CSA Biophen FIX.

Results

Acceptable recoveries for samples spiked at concentrations of 0.04, 0.08, 0.15, 0.61 and 1.22 IU/mL were achieved using CSA Biophen FIX (mean recovery 92% with the CN 3000 analyser and 108% with the STA R MAX analyser) and OSA STA CK Prest (mean recovery 92%, range 86%–111%). STA CK Prest showed FIX:C results comparable to Biophen FIX in both in vitro and post‐infusion samples.

Conclusions

STA CK Prest and Biophen FIX showed acceptable recoveries for N9‐GP, with FIX:C results in good agreement in both in vitro and post‐infusion samples. This supports the reliability of STA CK Prest for FIX:C measurement.

1. Introduction

Haemophilia B (HB) is a congenital X‐linked bleeding disorder characterised by reduced coagulation factor IX activity (FIX:C) level in plasma. It is caused by pathogenic variants in the F9 gene [1]. The severity is defined based on the degree of deficiency: severe (FIX:C <0.01 IU/mL), moderate (FIX:C 0.01–0.05 IU/mL) and mild (FIX:C 0.05–0.4 IU/mL) [2, 3]. The characteristic phenotype is the bleeding tendency, and the severity of bleeding correlates with the degree of the factor deficiency [1]. The classic manifestation is bleeding into the joints and muscles [1, 4]. Treatment options include factor replacement therapy, non‐replacement and gene therapies [5]. Replacement therapy with plasma‐derived (pdFIX) or recombinant FIX (rFIX) concentrates is the standard treatment for patients with severe HB either on demand or as regular prophylaxis [2, 3, 6]. Prophylaxis with factor concentrates reduces the frequency of joint and other bleeds because it changes the bleeding phenotype from a severe to a milder form [6, 7]. The standard half‐life of native FIX is 18–24 h and necessitates frequent intravenous administration [5, 8]. Therefore, recombinant factor concentrates were modified to extend their half‐life (EHL). FIX half‐life extension has been achieved by fusion with the fragment crystallizable (Fc) part of immunoglobulin G1 or albumin. Another technology is the covalent attachment of polyethylene glycol (PEGylation). The recombinant fusion proteins benefit from the endothelial neonatal Fc receptor recycling pathway, and PEGylation reduces renal filtration and clearance. The median half‐life of these products is three to five times longer than standard half‐life concentrates [5, 7, 9, 10].

Nonacog beta pegol (N9‐GP) is a serum‐free human recombinant factor IX with a 40 kDa polyethylene glycol (PEG) moiety selectively attached to one of the N‐glycans within the FIX activation peptide. This rFIX is produced in Chinese hamster ovary (CHO) K1 cells by recombinant DNA technology. Upon activation, the activation peptide, along with the PEG moiety, is cleaved off, resulting in activated rFIX (rFIXa) [11, 12]. In clinical studies, its half‐life was 93 h, which allows for the possibility of once–weekly prophylaxis [8, 11, 12, 13, 14, 15, 16].

Correct dosing with this product is assured by the accurate assignment of factor activity and relevant assessment of factor recovery after product administration [17]. Two main commercially available techniques for measuring FIX:C are applied: one‐stage clotting assay (OSA) and chromogenic substrate assay (CSA) [2, 18, 19]. OSA is based on dilution of the patient sample with FIX‐deficient plasma, followed by measurement of the activated partial thromboplastin time (APTT), with the final result determined by clot formation. In contrast, CSA is based on the generation of activated factor X (FXa), with the final result indicated by a colour change in a chromogenic substrate. Patient samples are strongly diluted, and the amount of FXa generated reflects the FIX:C in the sample [2, 18, 19].

Early studies demonstrated that certain assay methods tend to overestimate FIX:C of N9‐GP, whilst others result in its underestimation. The measurement with OSA is affected by PEGylation; however, CSA showed minimal PEG interference [20]. The variability appears to be dependent on the types of APTT reagents and reference standards used. There is a wide variety of APTT reagents, which differ in phospholipid type or concentration (synthetic, plant, or animal extracts) and activator type (e.g., ellagic acid, kaolin, silica of various types and polyphenols). The analysers, which differ in detection (mechanical or optical) and assay protocol, may also influence accurate measurement [2, 18]. Discrepancies between methods can also be explained by differences in calibration approaches: whilst the potency of rFIX concentrates is determined using calibrators based on factor concentrates, clinical laboratories calibrate assays against plasma standards [21]. According to the Summary of Product Characteristics, it is recommended to use CSA for monitoring treatment due to the interference of PEG in OSA with various APTT reagents. In the absence of CSA, using OSA with a qualified APTT reagent (e.g., Cephascreen) is recommended. [22]. If CSA or a qualified OSA is not available, it is recommended to use a reference laboratory [23]. The reagents containing silica particles can cause severe overestimation; therefore, these reagents should be avoided [2, 23, 24]. Rosén et al. have shown that overestimation with silica reagents (e.g., APTT‐SP, TriniCLOT APTT HS, STA APTT A and Pathromtin SL) may occur since the PEG moiety mediates N9‐GP colocalisation with its activators (FXIa and prekallikrein) on the surface of silica particles, which leads to conversion of FIX to FIXa during the activation phase, in the absence of calcium [25]. This effect was not observed with the silica‐based reagent SynthASil, possibly due to the specific form of its colloidal silica particles. It is likely that N9‐GP does not interact with this type of silica, or that the low silica concentration in the reagent limits such interaction [18, 25].

As reported by Bowyer et al., N9‐GP can be accurately measured using CSA Rox FIX or Biophen FIX and OSA with SynthAFax and DG‐APTT Synth. The reagents SynthASil, Actin, Actin FS and Actin FSL lead to underestimation by 50%–70% [8]. This could be explained by the slower activation of N9‐GP, which apparently poses a greater steric challenge after PEG conjugation [26]. There are discrepant data regarding the reagent STA CK Prest. Some studies describe that this reagent caused underestimation [18, 26, 27], but another overestimation [28].

The recommendations of The World Federation of Haemophilia (WFH), the French Study Group on the Biology of Haemorrhagic Diseases (the BIMHO group) and The United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) for the use of specific methods and reagents are summarised in Table 1 [1, 24, 29]. The WFH recommends the use of CSA or OSA with validated reagents (SynthAFax or STA Cephascreen), calibrated with a plasma standard traceable to World Health Organization (WHO) International Standard (IS) [1].

TABLE 1.

Overview of reagent‐specific recommendations from WFH, UKHCDO, and BIMHO.

	WFH [1]	UKHCDO [24]	BIMHO [29]
APTT reagent
Silica
STA PTT‐A	NM	Unsuitable	NM
Pathromtin SL	NM	Unsuitable	Unsuitable
SynthASil	NM	Unsuitable	Unsuitable
APTT‐SP	NM	Unsuitable	Unsuitable
Triniclot Auto	NM	NM	NM
Triniclot HS	NM	NM	NM
DG‐Synth	NM	Suitable	Suitable
Ellagic acid
Actin	NM	Unsuitable	Unsuitable
Actin FS	NM	Unsuitable	Unsuitable
Actin FSL	NM	Unsuitable	Unsuitable
SynthAFax	Suitable	Suitable	Suitable
Kaolin
STA CK Prest	NM	NM	NM
Polyphenols
STA Cephascreen	Suitable	Suitable	Suitable
CSA
Biophen FIX	Suitable	Suitable	Suitable
Rox FIX	Suitable	Suitable	Suitable

Abbreviation: NM, not mentioned.

Although recent studies have identified CSA and OSA using STA Cephascreen, DG‐APTT Synth, or SynthAFax reagents as the preferred assays for measuring FIX:C levels of N9‐GP [8, 24, 29, 30], it should be noted that STA Cephascreen and DG‐APTT Synth are no longer commercially available. Given that CSA may be less practical for urgent monitoring, such as in perioperative management, we conducted this study to assess the suitability of STA CK Prest.

The aim of this study was therefore to evaluate the recovery of N9‐GP using four different APTT reagents with particular emphasis on STA CK Prest and one CSA, across different analysers. The assessment was conducted on both in vitro spiked samples and on post‐infusion patient samples.

2. Materials and Methods

2.1. In Vitro Sample Preparation

In vitro samples were prepared by diluting N9‐GP (Refixia 500 IU, Novo Nordisk, Denmark) in commercially available FIX‐deficient plasma (STA Deficient IX, Diagnostica Stago, France) to achieve nominal FIX:C concentrations of 0.04, 0.08, 0.15, 0.61 and 1.22 IU/mL. Spiked concentrations were based on the manufacturer's labelled potency. Aliquots were prepared for each concentration. The initial measurements of FIX:C by OSA and CSA were performed immediately after sample preparation, whilst the remaining aliquots were stored at –80°C and analysed on subsequent days. Prior to analysis, frozen samples were thawed in a water bath at 37°C for 5 min.

2.2. Post‐Infusion Patient Samples

Blood samples were collected from six patients with no history of inhibitors at various time points after N9‐GP infusion during routine follow‐up visits. Ethical approval was obtained from the local ethics committee (28‐080323/EK). All samples were collected and processed in accordance with the Declaration of Helsinki. No additional blood sampling was performed beyond routine clinical care.

Blood was drawn into 0.106 M sodium citrate tubes. For plasma analysis, the tubes were centrifuged at 2500 g for 15 min at room temperature. The plasma was frozen and stored at –80°C until analysis. Prior to analysis, frozen samples were thawed in a 37°C water bath for 5 min.

2.3. OSAs

In vitro and post‐infusion samples were measured using the STA R MAX (Diagnostica Stago, France), the Atellica COAG 360 (Siemens, Germany) and the CN 3000 (Sysmex, Japan). We used four APTT reagents with different activators: ellagic acid (Actin FS and Actin FSL, Siemens, Germany), kaolin (STA CK Prest, Diagnostica Stago, France) and silicon dioxide (Pathromtin SL, Siemens, Germany). The methods were calibrated using assay‐specific commercial plasma standards traceable to the WHO IS: STA Unicalibrator (Diagnostica Stago, France) or Standard Human Plasma (Siemens, Germany). The deficient FIX plasma was also assay specific: STA Immunodef IX (Diagnostica Stago, France) for STA CK Prest and FIX deficient (Siemens, Germany) for Actin FS, Actin FSL and Pathromtin SL. Calibration curves were validated using internal controls at normal and pathological levels, specific to the analyser and assay: STA System Control N+P (Diagnostica Stago, France) or Control N and Control P (Siemens, Germany). In vitro samples were measured once using Actin FS, Actin FSL and Pathromtin SL on the Atellica COAG 360 and CN 3000 analysers, due to their known tendency to under‐ or overestimate FIX:C levels. In contrast, STA CK Prest was assessed in triplicate across three independent experiments using the STA R MAX analyser.

2.4. CSA

FIX:C in both in vitro and post‐infusion samples was measured using the CSA kit Biophen FIX (Hyphen Biomed, France). The assay was performed on two analytical platforms: STA R MAX (Diagnostica Stago, France) using the calibration curves with plasma standard STA Unicalibrator (Diagnostica Stago, France), and CN 3000 (Sysmex, Japan) using Biophen Plasma Calibrator (Hyphen Biomed, France). Both plasma standards are traceable to the WHO IS. Calibration curves were validated using two levels of internal quality control: STA System Control N+P (Diagnostica Stago, France) or Biophen Plasma Control (Hyphen Biomed, France), respectively. In vitro samples were analysed in triplicate across three independent experiments.

2.5. Statistical Analysis

The recovery percentage was calculated for each spiked sample as follows:

$$\frac{\mathrm{Mean}\mathrm{measured}\mathrm{FIX}:\mathrm{C}}{\mathrm{Target}\mathrm{FIX}:\mathrm{C}}\times 100$$

In accordance with the UKHCDO guidelines, a recovery within 100 ± 20% was considered acceptable for samples with FIX levels above 0.3 IU/mL, and within 100 ± 30% for levels between 0.1 and 0.3 IU/mL [24].

The FIX:C results were compared using Bland–Altman plots and linear regression analysis. Statistical analyses were performed using Microsoft Excel and GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Assays That Underestimated or Overestimated

Recovery of N9‐GP was markedly underestimated across all concentrations when measured by OSA with Actin FS and Actin FSL. For Actin FS, recovery ranged from 31%–42% on the Atellica COAG 360 and 23%–34% on the CN 3000. For Actin FSL, recovery was 47%–61% on the Atellica COAG 360 and 46%–60% on the CN 3000. These results show comparable underestimation of FIX:C across both analysers (Figure 1).

FIGURE 1.

Recovery of N9‐GP measured by different assays and analysers. CSA Biophen1 was performed on the STA R MAX, and Biophen2 on the CN 3000. OSA Actin FS1 and Actin FSL1 measurements were performed on the Atellica COAG 360, whilst Actin FS2 and Actin FSL2 were performed on the CN 3000. The solid line indicates 100% recovery. The dashed lines represent recovery within 100 ± 20%, which was considered acceptable for samples with FIX levels above 0.3 IU/mL, whereas the dotted lines represent recovery within 100 ± 30%, acceptable for samples with FIX levels between 0.1 and 0.3 IU/mL.

In contrast, OSA with Pathromtin SL markedly overestimated FIX:C. Mean recovery reached 1727% at 0.04–0.15 IU/mL, whilst at higher concentrations, FIX:C exceeded the upper measurement limit (>4.0 IU/mL), preventing recovery calculation.

3.2. Assay Demonstrating Acceptable Recovery

Acceptable recovery was observed using CSA Biophen FIX regardless of the measurement system. The mean recovery on the CN 3000 analyser was 92%, with values ranging from 83% to 104%. Using the STA R MAX analyser, the mean recovery was 108%, with values ranging from 102% to 116%. Data are presented in Figure 1 and Table 2. Similarly, acceptable recovery was achieved using OSA with STA CK Prest. The mean recovery was 92%, ranging from 86% in high‐concentration samples to 111% in samples with very low N9‐GP concentrations. The results are shown in Figure 1 and Table 2.

TABLE 2.

Mean recovery and precision for each N9‐GP level of the OSA with STA CK Prest, and CSA Biophen FIX using different analysers.

	STA CK Prest/STA R MAX	Biophen FIX/STA R MAX	Biophen FIX/CN 3000
Accuracy	Mean % recovery	Mean % recovery	Mean % recovery
0.04 IU/mL	111	102	83
0.08 IU/mL	90	111	88
0.15 IU/mL	88	116	89
0.61 IU/mL	87	110	104
1.22 IU/mL	86	103	94

Precision	%CV	%CV	%CV
0.04 IU/mL	9.63	3.21	2.24
0.08 IU/mL	6.05	1.47	6.33
0.15 IU/mL	6.76	3.65	2.61
0.61 IU/mL	6.24	1.87	5.8
1.22 IU/mL	2.16	3.09	4.06

Abbreviation: %CV, percentage coefficient of variation.

Low coefficients of variation (CVs), all below 10%, were observed across the tested concentrations in each spiked sample, indicating good reproducibility of the methods. These results also demonstrate that there were no significant differences between the measurements obtained from fresh samples and those from samples that were frozen and subsequently thawed, indicating that freezing does not adversely affect the assay results (Table 2).

3.3. Comparison of Biophen FIX Assay Results Performed on STA R MAX and CN 3000 Analysers

The mean recovery results of the FIX assay Biophen FIX using the STA R MAX and CN 3000 analysers are summarised in Table 2. FIX:C obtained by STA R MAX was slightly higher than that obtained by CN 3000, but linear regression showed excellent linearity (r = 0.997) (Figure 2). The observed difference may be attributed to variations between the analysers and the use of different plasma calibrators.

FIGURE 2.

Linear regression analysis of Biophen FIX results. FIX:C results of spiked samples measured on the STA R MAX and CN 3000 analysers show a strong correlation (r = 0.997). The regression line (solid) has a slope of 0.91 (95% CI: 0.87–0.95). A dashed diagonal line representing the line of identity (y = x) is included for reference. The slope below 1 suggests that the CN 3000 analyser tends to yield slightly lower recovery values compared to the STA R MAX.

3.4. Comparison of Results Obtained by OSA STA CK Prest and CSA Biophen FIX

Minor differences were observed between Biophen FIX (on both analysers) and OSA STA CK Prest. Agreement between the methods was assessed using two Bland‐Altman plots, which suggest that discrepancies between methods increased at higher FIX:C levels. Despite a slight negative bias of the OSA method compared to the CSA, overall agreement remained within clinically acceptable limits (see Figure S1a,b).

3.5. In Vitro Spiked and Post‐Infusion Samples

An analysis of N9‐GP activity in spiked and post‐infusion samples using CSA Biophen FIX (on both STA R MAX and CN 3000 analysers) and OSA with STA CK Prest showed generally minor differences between assays at low FIX:C levels, but slightly greater discrepancies at higher concentrations. The results were compared using a Bland‐Altman plot, which suggests that spiked and post‐infusion samples behave similarly (Figure 3).

FIGURE 3.

Bland‐Altman plot of in vitro spiked and post‐infusion samples. The difference in N9‐GP activity measurements between CSA Biophen FIX and OSA STA CK Prest was evaluated for both spiked and post‐infusion samples. For each sample, the average of FIX:C measured by the two methods was plotted against the difference between the values obtained. The values of in vitro spiked samples are expressed as the mean of three measurements for each sample and method.

4. Discussion

Modifications of factor IX concentrates are designed to reduce their clearance from the patient´s circulation; however, they also introduce challenges in laboratory monitoring, which is essential to ensure optimal therapy and to minimise the risk of both underdosing and overdosing. For this purpose, clinical laboratories use commercially available OSA and CSA, with OSA being more commonly used in routine practice. According to previous studies, some OSA may significantly underestimate or overestimate the activity of N9‐GP, mainly due to the choice of APTT reagents. N9‐GP can be accurately measured using CSA Biophen FIX and Rox FIX or using OSA with APTT reagents SynthAFax, STA Cephascreen, or DG‐APTT Synth [22, 24, 29].

Since neither SynthAFax—which cannot be used on our analysers (Stago, Siemens and Sysmex) due to in vitro diagnostics regulation (IVDR)—nor STA Cephascreen and DG‐APTT Synth (which are no longer commercially available), nor CSA—which is less practical for urgent monitoring, such as in perioperative settings—was suitable, we aimed to evaluate the suitability of STA CK Prest.

We confirmed that Actin FS, Actin FSL and Pathromtin SL are unsuitable for monitoring of N9‐GP treatment. OSA with Actin FS and Actin FSL was substantially underestimated by approximately 45%–70%, whereas Pathromtin SL was markedly overestimated due to its silica content. These findings are consistent with previously published data [8, 31, 32, 33].

Acceptable recovery was observed with CSA Biophen FIX, as expected. The FIX:C results from both tested analysers were comparable. However, recovery was slightly higher—particularly at low concentration levels—when using the STA R MAX analyser compared to the CN 3000. This difference may be attributed to variations between the analysers and the use of different plasma calibrators. Another method that demonstrated accurate performance was OSA with STA CK Prest. The mean recovery was 92% (range 86%–111%), and its results showed good agreement with the Biophen FIX assay, indicating that STA CK Prest may be a suitable option for the routine monitoring of N9‐GP. This supports its potential utility in clinical laboratories, particularly in situations where CSA is less feasible due to cost or reagent stability. However, this finding contrasts with earlier publications, reflecting a degree of inconsistency within the existing literature.

Persson et al. demonstrated that certain APTT reagents (Actin FS, SynthASil, STA CK Prest) lead to slower activation of N9‐GP as a result of FXIa being adsorbed onto surfaces during the contact activation phase, which reduces its availability for activating N9‐GP [26, 33]. In another study, Nederlof et al. evaluated the performance of assays using in vitro‐spiked samples containing N9‐GP at concentrations of 6 and 60 IU/dL, as a part of an external quality assessment programme [27]. Unexpectedly, an overestimation of FIX:C was observed in both spiked samples when measured with SynthAFax. In both samples, the group of reagents—Stago Cephalin, Kaolin and STA CK Prest—yielded underestimated values, accompanied by high coefficients of variation. Moreover, considerable inter‐assay variability was noted for rFIX‐EHL concentrates across both one‐stage and chromogenic methods [27].

In contrast, Duboscq et al. observed differing results with STA CK Prest, further emphasising the inconsistencies found in the literature [28]. The study evaluated in vitro‐spiked samples at four concentration levels across ten APTT reagents. Measurements with STA CK Prest on the STA Compact analyser, calibrated with plasma standard, resulted in an overestimation of FIX:C. [28].

The comparison of findings from different studies is complicated by variations in reagents, analysers and calibration standards used. The same reagent can be used on different analysers with varying assay protocols and detection systems (optical or mechanical). Incubation time can also play an important role, as the activator contained in the APTT reagent leads to the activation of FXI, which activates FIX. Some studies use the WHO FIX IS as a calibration reference, whereas others employ plasma standards traceable to the WHO IS. In addition, the preparation of spiked samples and the precision of pipetting and dilution procedures are critical factors that may contribute to variability of the results.

In our study, we confirmed that STA CK Prest achieves acceptable recovery and demonstrates consistent performance in both spiked and post‐infusion patient samples. The observation that the coefficient of variation of spiked samples did not exceed 10% across all activity levels provides further evidence of the high precision and robustness of the assay using STA CK Prest. This result substantially meets the WFH's recommended reproducibility criterion of <15% [1]. When comparing its results to CSA—recommended by multiple clinical guidelines—we observed good agreement between these two methods. Similar behaviour was evident in both in vitro and patient samples, supporting the findings of Sørensen et al. [34].

However, our study has some limitations. For in vitro sample testing, we used commercially immunodepleted plasma rather than congenital FIX‐deficient plasma, and OSA methods with Actin FS, Actin FS and Pathromtin SL were measured only once. Another limitation is that we evaluated only Siemens and Stago systems and were unable to include products from other manufacturers.

5. Conclusions

The results of this study support the suitability of the following reagent–instrument systems for monitoring N9‐GP: OSA calibrated with a plasma standard, utilising STA CK Prest and STA Immunodeficient Plasma on the STA R MAX analyser; and CSA Biophen FIX performed on either the STA R MAX or CN 3000 analysers, both calibrated using a plasma standard. However, it should be noted that validation within individual clinical laboratories remains essential.

Author Contributions

Marie Prudková designed and performed the experiments, analysed data and drafted the manuscript. Dagmar Chytrá performed the experiments. Petr Smejkal contributed patient samples and revised the manuscript. Jiřina Zavřelová revised the manuscript. Gabriela Romanová contributed patient samples and revised the manuscript. Miroslav Penka revised the manuscript. Alena Buliková revised the manuscript.

Funding

This work was supported by the Ministry of Health of the Czech Republic—conceptual development of research organisation (FNBr, 65269705), and by Masaryk University Brno (grant no. MUNI/A/1685/2024).

Ethics Statement

This study was conducted in accordance with the Declaration of Helsinki and the ethics committee of University Hospital Brno (28‐080323/EK).

Conflicts of Interest

Marie Prudková received speaker fees from Takeda and Sobi. Petr Smejkal received speaker and/or consultancy fees from Novo Nordisk, Roche, Takeda, Sobi and CSL Behring. Gabriela Romanová received a speaker and/or consultancy fee from Sobi. The other authors declare no conflicts of interest.

Supporting information

Supplementary Figure S1a: Bland‐Altman analysis of FIX:C in spiked samples. For each sample, the mean FIX:C obtained from CSA (Biophen FIX on CN 3000) and OSA with STA CK Prest (STA R MAX) was plotted against the difference in values between the two methods. The mean difference was −0.053 IU/mL, with 95% limits of agreement (mean ± 1.96 SD) ranging from −0.185 to 0.08 IU/mL. Supplementary Figure S1b: Bland‐Altman analysis of FIX:C in spiked samples. For each sample, the mean FIX:C obtained from CSA (Biophen FIX on STA R MAX) and OSA with STA CK Prest (STA R MAX) was plotted against the difference in values between the two methods. The mean difference was −0.075 IU/mL, with 95% limits of agreement (mean ± 1.96 SD) ranging from −0.233 to 0.08 IU/mL.

Prudková M., Smejkal P., Chytrá D., et al. “Monitoring of Nonacog Beta Pegol: One‐Stage Clotting Assay With Kaolin Reagent as a Practical Alternative to Chromogenic Methods.” Haemophilia 32, no. 1 (2026): 284–291. 10.1111/hae.70168

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.